durable therapeutic efﬁcacy utilizing combinatorial...

Cancer Therapy: PreclinicalSee related commentary by Castro et al., p. 5147

Durable Therapeutic Efficacy Utilizing CombinatorialBlockade against IDO, CTLA-4, and PD-L1 in Mice with BrainTumors

Derek A. Wainwright, Alan L. Chang, Mahua Dey, Irina V. Balyasnikova, Chung Kwon Kim, Alex Tobias,Yu Cheng, Julius W. Kim, Jian Qiao, Lingjiao Zhang, Yu Han, and Maciej S. Lesniak

AbstractPurpose: Glioblastoma (GBM) is the most common form of malignant glioma in adults. Although

protected byboth theblood–brain andblood–tumorbarriers,GBMs are actively infiltrated byT cells. Previous

workhas shownthat IDO,CTLA-4, andPD-L1aredominantmolecularparticipants in the suppressionofGBM

immunity. This includes IDO-mediated regulatory T-cell (Treg; CD4þCD25þFoxP3þ) accumulation, the

interaction of T-cell–expressed, CTLA-4, with dendritic cell-expressed, CD80, as well as the interaction of

tumor- and/or macrophage-expressed, PD-L1, with T-cell–expressed, PD-1. The individual inhibition of each

pathway has been shown to increase survival in the context of experimental GBM. However, the impact of

simultaneously targeting all three pathways in brain tumors has been left unanswered.

Experimental Design and Results: In this report, we demonstrate that, when dually challenged, IDO-

deficient tumors provide a selectively competitive survival advantage against IDO-competent tumors. Next,

we provide novel observations regarding tryptophan catabolic enzyme expression, before showing that the

therapeutic inhibition of IDO, CTLA-4, and PD-L1 in amousemodel of well-established gliomamaximally

decreases tumor-infiltrating Tregs, coincident with a significant increase in T-cell–mediated long-term

survival. In fact, 100% of mice bearing intracranial tumors were long-term survivors following triple

combination therapy. The expression and/or frequency of T cell expressed CD44, CTLA-4, PD-1, and IFN-gdepended on timing after immunotherapeutic administration.

Conclusions: Collectively, these data provide strong preclinical evidence that combinatorially targeting

immunosuppression inmalignant glioma is a strategy that hashighpotential value for future clinical trials in

patients with GBM. Clin Cancer Res; 20(20); 5290–301. �2014 AACR.

IntroductionGlioblastoma multiforme (GBM) is the most common

malignant brain tumor in adults, with more than 50,000patients currently living with the disease (1, 2). Althoughpatients with GBM may undergo aggressive surgical resec-tion, irradiation, and chemotherapy, the overall survivalremains at only 14.6 months after diagnosis (3). GBM hasbeen genomically stratified into four major subgroups: (i)neural, (ii) proneural, (iii) classical, and (iv) mesenchymal(4). Different subtypes arise due to distinct pro-oncogenicevents, possess novel response rates to therapy, and show

diverse patterns of T-cell infiltration (5). Given the hetero-geneity of GBM, it has been a challenge to find therapeu-tically targetable genes that are expressed by all four geno-mic subtypes.

Indoleamine 2, 3 dioxygenase 1 (IDO) is a tryptophancatabolic enzyme not normally expressed in the CNS paren-chyma at appreciable levels (6), but is induced in 90% ofpatients with GBM (7). Moreover, higher IDO expression ismore often observed in GBM when compared with low-grade glioma (8), suggesting that the degree of transforma-tion may play a role in IDO expression levels. Moreover,patients withGBMwith higher IDO expression levels exhib-it a shorter overall survival. Recentwork fromour laboratoryhas identified that IDO expression by brain tumor cells,rather thanother cells capableof expressing IDO, is criticallyimportant in tumor-induced suppression of the antitumorresponse (9). This was an unexpected finding given that inperipheral tumor models, IDO expressed by dendritic cells(DC) appears to play a critical role in suppressing theantitumor immune response (10–12).

Immunosuppression in glioblastoma is a characteristichallmark associated with the recruitment of myeloid-derived suppressor cells (13, 14), increased interleukin-10

Authors' Affiliation: The Brain Tumor Center, The University of ChicagoPritzker School of Medicine, Chicago, Illinois

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Maciej S. Lesniak, The University of ChicagoPritzker School ofMedicine, 5841 S.Maryland Avenue, MC3026, Chicago,IL 60637. Phone: 773-834-4757; Fax: 773-834-2608; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0514

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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and TGF-b (15–17), as well as the accumulation of regula-tory T cells (Treg; CD4þCD25þFoxP3þ; refs. 18, 19). Thelatter component is a potently immunosuppressive subsetthat in GBM is characterized by the constitutively highlevel of CTLA-4, GITR and is predominantly representedas being thymus derived (20). On the basis of previousstudies in peripheral tumor models demonstrating theimpact of IDO on Treg activation (21), expansion (22)and/or recruitment (23), we recently asked whether therewas a dominant cell type that required IDO to regulateTreg levels in brain tumors (9). Our original hypothesisassumed that DC-expressed IDO would be required,based on previous work showing their role in Treg mod-ulation (10, 21, 24). However, upon more careful exam-ination, while IDO-expressing DCs control Treg expan-sion, this mechanism depends on the conversion ofCD4þCD25� non-Tregs into CD4þCD25þ(FoxP3þ) Tregs(25–27). Given that glioma-resident Tregs are primarilythymus derived (20), this likely explains why IDO�/�DCfailed to impact Treg levels in brain tumors.Here, we first extend our previous observations by

determining the immunodominance of IDO-competentand IDO-deficient malignant glioma, ultimately revealingthat timing of tumor implantation plays a significant rolein tumor rejection. Next, we determined the therapeuticimpact of inhibiting IDO, alone, or when combined withthe current standard-of-care therapy, temozolomide(Temodar), unexpectedly revealing that the enzymaticinhibition of IDO does not play a crucial role in signif-icantly increasing overall survival in malignant glioma.Importantly, we tested the individual and combinedimpact of inhibiting CTLA-4, PD-L1, and IDO in modelsof established glioma, demonstrating a robust decrease in

tumor-resident Tregs concurrent with increased survivalwhen all three targets were inhibited simultaneously.However, when we tested the impact of a similar strategyin an aggressive intracranial melanoma model, only amodest effect on survival was observed. Collectively,these data significantly enhance our understanding oftherapeutically targetable immunosuppressive pathwaysactive in brain tumors.

Materials and MethodsMice and cell lines

C57BL/6 (wild-type; Cat# 000664), IDO�/� (Cat#005897), Rag1�/� (Cat# 002216), and OT-II (Cat#004194) mice were obtained from Jackson Laboratories,maintained in the University of Chicago Carlson BarrierFacility and intracranially injected between the ages of 6and 8 weeks. All mouse strains used in the includedstudies were on the C57BL/6 background and were pro-vided autoclaved food pellets and water ad libitum. Allsurgical procedures were completed in accordance withNIH guidelines on the care and use of laboratory animalsfor research purposes. All studies performed on mice wereapproved by the Institutional Animal Care and Use Com-mittee of the University of Chicago (Chicago, IL). Micewere euthanized by cervical dislocation. GL261 and B16-F10 cells were obtained from the NCI Frederick NationalTumor Repository Lab (no authentication of the cell lineswas conducted) and cultured in Dulbecco modified Eaglemedium supplemented with 10% fetal calf serum as wellas streptomycin (100 mg/mL) and penicillin (100 U/mL)at 37�C in a humidified atmosphere of 95% air/5% CO2.All cell culture products were purchased from GibcoInvitrogen.

Mouse orthotopic intracranial injection modelAdetailed description canbe found in the Supplementary

Materials and Methods section.

Reagents and treatmentsAdetailed description canbe found in the Supplementary


Western blottingAdetailed description canbe found in the Supplementary


Flow cytometry and T-cell stimulationAdetailed description canbe found in the Supplementary


Statistical analysisData were analyzed using Prism 4.0 software (GraphPad

Software). Experiments were repeated at least two timeseach. Data are represented as the mean� SEM for all figurepanels inwhich error bars are shown. The P values representANOVA for groups of 3 or more, whereas two-tailedunpaired Student t tests were used for paired groups.

Translational RelevanceThe use of agents that reverse immunosuppression in

tumors is an approach that is gaining clinical traction.Recently, Wolchok and colleagues (2013) demonstratedthat the combination of immune checkpoint blockadeinhibitors (via CTLA-4 and PD-1 mAb) resulted in anobjective response in patientswith end-stagemelanoma.However, whether a similar type of approach will beeffective in patients with intracranial tumors hasremained an elusive question. Here, we first extendedour previous findings by investigating the role of IDO1in brain tumors, followed by the presentation of pre-clinical findings demonstrating a highly effective thera-peutic strategy that simultaneously targets immunecheckpoints and tryptophan catabolism inbrain tumors.Given the current interest for exploring clinicaltrials utilizing CTLA-4, PD-(L)1, and/or IDO blockadein patients with brain tumor, these results serve as animportant proof-of-concept supporting the future pur-suit of this strategy in patients with incurableglioblastoma.

IDO, CTLA-4, PD-L1 Synergistic Blockade in Brain Tumors

www.aacrjournals.org Clin Cancer Res; 20(20) October 15, 2014 5291




A P value of less than 0.05 was considered statisticallysignificant.

ResultsThe role of IDO and antigen specificity in gliomaimmunity

The genetic ablation of IDO in glioma cells results in thespontaneous rejection of brain tumors mediated by T cells(9). Previous work demonstrating that the majority ofpatient GBM specimens are >50% positive for IDO (7)suggests that this tryptophan catabolic enzyme tonicallymaintains suppression of the antitumor response. To deter-mine the minimum number of IDO-deficient cells ina brain tumor required to induce tumor rejection, wemixedIDO-competent and IDO-deficient GL261 cells at variousratios and tested the effects on survival in IDO1-deficient(IDO�/�) mice. As shown in Fig. 1A, 100% of glioma-bearing mice with IDO-competent (Vc) tumor cellsdiedwith a median overall survival of 24 days. In contrast,glioma-bearing mice with tumors mixed with IDO-com-petent and -deficient (IDOkd) tumor cells at 3:1, 1:1, or1:3, resulted in 40% of mice surviving for up to 150 days

(P < 0.05, <0.01, and <0.001, respectively). However,even with the survival advantage conveyed by the differ-ent ratios of IDO-deficient glioma cells, it was still overalllower when compared with the group of mice intracra-nially injected with IDO-deficient cells, alone, whichresulted in 75% of mice surviving for up to 150 daysafter intracranial injection (P < 0.001).

To determine the nature and strength of the antitumorresponse induced by IDO-deficient glioma cells, we estab-lished IDO-competent and/or IDO-deficient tumor cells inboth cerebral hemispheres of IDO�/�mouse brain to betterunderstand IDO-dependent glioma-induced immunodo-minance. As shown in Fig. 1B, when mice were simulta-neously injected IDO-competent cells on both sides of themouse brain, 100% of mice died with a median survival of15.5 days after intracranial injection. Interestingly, whenmice were simultaneously injected IDO-competent and-deficient cells in opposite cerebral hemispheres, 100% ofmice died with a median survival of 22 days. This was incontrast with the survival benefit imparted when IDO-deficient glioma cells were intracranially injected into bothcerebral hemispheres, resulting in 80%ofmice surviving up

Figure 1. The rejection of IDO-competent and -deficient brain tumors is context-dependent. A, survival analysis of indoleamine 2,3 dioxygenase knockout(IDO�/�) mice intracranially injected (i.c.) with a total of 4 � 105 GL261 cells transduced with -scrambled shRNA (vector control, Vc), -shRNA specific to IDO(IDO knockdown, IDOkd), or mixed (Vcþ IDOkd) at different ratios of cells. �,P < 0.05; ��,P < 0.01; ��,P < 0.001 (n¼ 5–11/group). B, survival analysis of IDO�/�

mice i.c. injectedwith 4� 105 Vc or IDOkdGL261 cells in the right (right) and left (left) cerebral hemispheres, simultaneously [Day 0 (D0)], or 4� 105 IDOkdGL261were i.c. injected in the right hemisphere (D0), followed by an intracranial injection of 4� 105 Vc GL261 cells at 7 or 32 days after i.c. (D7 or D32, respectively;n ¼ 4–8/group). ��, P < 0.01. C, survival analysis of wild-type (WT) or OT-II (CD4þ T cells specific to chicken ovalbumin 323-339 I-Ab) mice i.c. injectedwith 4� 105 unmodified GL261 (n¼ 5–7/group). D, the frequency of CD4þFoxP3þ Tregs (left) and frequency of Tregs bearing the Va2 receptor isolated frombrain tumors derived from unmodifiedGL261 cells analyzed at 3 weeks after i.c. Tregs were initially gated on CD3 and CD4. Bar graphs are shown asmean�SEM (n ¼ 4–5 mice/group). E, survival analysis of WT or OT-II mice i.c. injected with 4 � 105 Vc or IDOkd GL261 cells (n ¼ 3 mice/group). For survivalexperiments, mice were analyzed for up to 150 days and results reflect the data from two independent experiments.

Wainwright et al.

Clin Cancer Res; 20(20) October 15, 2014 Clinical Cancer Research5292




to 150 days after intracranial injection (P < 0.001). Whentaking into consideration the results fromFig. 1A, these datacollectively suggest that themicroenvironmentwithin IDO-competent gliomas is sufficient to induce a coordinatedimmunosuppressive response that overcomes the antitu-mor response elicited by completely IDO-deficient satellitetumors in the brain. We next tested whether the priorestablishment of IDO-deficient tumors would be sufficientfor rejecting IDO-competent tumors. In mice already bear-ing IDO-deficient tumors, when IDO-competent gliomacells were implanted early (i.e., 7 days after intracranialinjection), 100% of mice survived, whereas a later rechal-lenge with IDO-competent glioma cells (i.e., 32 days afterintracranial injection) led to only 40% of mice surviving(P<0.001, respectively). Thesedatahighlight the contextualnature of the antitumor immune response mediated byIDO-deficient glioma cells and suggest that timingof rechal-lenge plays a critical role with regard to overcoming tumor-induced immunosuppression and consequent effects onsurvival.Given the potent effects of glioma cell-specific IDO-defi-

ciency on the induction of productive tumor immunity,we next wondered whether this effect required antigenspecificity or whether the nonspecific presence of CD4þ Tcells was sufficient for increasing overall survival. Asexpected, Fig. 1C shows that neither wild-type (WT) norOT-II (antigen restricted CD4þ T cells to chicken ovalbu-min) mice mounted a long-term survival effect when intra-cranially injected with normal (IDO-competent) gliomacells. Notably, bothWT andOT-IImice showed a significantaccumulationof Tregs in thebrain, althoughOT-IImice hadrelatively decreased levels (P < 0.01).WhenOT-IImice were

intracranially injected with IDO-competent or -deficientglioma cells, neither group could mount a long-term (i.e., up to 150 days after intracranial injection) survivalresponse. These data suggest that antigen-specific CD4þ Tcells are required for mediating the antitumor response inthe context of IDO-deficient brain tumors.

IDO expression and combinatorial targeting withtemozolomide in glioma

Previous work from peripheral tumor models has shownthat inhibiting IDO with the well-characterized molecularagent, 1-methyltryptophan (1-MT), requires the addition ofa chemotherapeutic agent to provide a significant antitumorresponse (10). To address this, we hypothesized that 1-MTwould synergize with temozolomide, the current standard-of-care chemotherapeutic agent for patients with glioblas-toma, to achieve a synergistic antitumor-mediated survivalbenefit. As shown in Fig. 2A and B, neither L1-MT nor D1-MT in glioma-bearing mice significantly impacted overallsurvival,whencomparedwith control untreatedmicewith amedian survival of 24.5 days after intracranial injection. Incontrast, the treatment of mice with temozolomide, alone,led to an increased median survival of 37.5 days afterintracranial injection. (P < 0.01). To our surprise, neitherthe additionof L1-MTnorD1-MT increased survival further,versus treatmentwith temozolomide, alone.However, therewas a small but significant survival advantage when D1-MTwas coupled with temozolomide, compared with L1-MTwith temozolomide, reflectedby themedianoverall survivalof 46 versus 35 days after intracranial injection (P < 0.05).

Given the surprisingly low level of efficacy upon treat-ment with 1-MT and temozolomide, we hypothesized that

Figure 2. Impact of IDO inhibitionand tryptophan catabolic enzymeexpression in brain tumors.A, timeline illustrating when oral(through the drinking water) 1-MT(2 mg/mL) and intraperitoneallyinjected temozolomide (TMZ) wasadministered to mice afterintracranial injection (i.c.) of 4� 105

GL261 cells. B, survival analysisrepresents mice that received theL- or D-stereoisomer of 1-MT, TMZ,or a combination of each 1-MTstereoisomer with TMZ (n ¼6/group). �, P < 0.05; ��, P < 0.01.C, representative Western blotsand (D) relative quantitativeanalysis for IDO1, IDO2, TDO, andchicken b-actin in GL261 cell-based glioma lysates isolated at1 and 3 weeks after i.c. in WT,IDO�/�, and Rag1�/� mice(n ¼ 3/group). �, P < 0.05;��, P < 0.01.






additional tryptophan catabolic pathways are present in thecontext of glioma, given the recent evidence that IDO2 andTDO also play a role in tumor immunity (28, 29). Wequantified the expression of IDO1, IDO2, and TDO fromnormal (IDO-competent) GL261 cell-based tumor lysatesat 1 and 3 weeks after intracranial injection from WT,IDO�/�, and Rag1�/� (lack functional T and B cells) micein Fig. 2C andD.Not surprisingly, when comparedwithWTmice, IDO expression was unchanged in both IDO�/� andRag1�/� mice, concordant with data showing that tumorsrequire IDO to suppress tumor immunity, as well as infer-ring that the tumor is a primary source of IDO expression.Interestingly, IDO2 was expressed in WT mice bearingglioma and that expression was enhanced by peripheralIDO deficiency (P < 0.05) as well as the temporal-sensitivityto the absence of T cells (P < 0.05). Equally, if not moreintriguing was the expression of TDO in WT and IDO�/�

mice that was potently decreased by the absence of func-tional T cells (P < 0.05). As far as we know, this is the firstreported observation that the immune systemplays a role in

regulating TDO expression in tumors. Collectively, thesedata suggest that due to the presence of IDO2 and TDO inglioma, therapeutic intervention against all three mamma-lian tryptophan catabolic enzymes may require simulta-neous blockade to significantly impact brain tumorimmunity.

A durable survival benefit after CTLA-4/PD-L1/IDOblockade in glioma

As an alternative to the combination of 1-MT with che-motherapy, we decided to pursue an approach that utilized1-MT with CTLA-4 and PD-L1 blockade, instead, given therecent clinical success for end-stage patientswithmelanomaadministered with CTLA-4 and PD-1 monoclonal antibo-dies (mAb; ref. 30). As shown in Fig. 3A and B, 100% ofuntreated mice died with a median survival of 29 days afterintracranial injection. In contrast, 40%, 60%, and 90% ofmice treated with CTLA-4 mAb, PD-L1 mAb, and coadmi-nistered CTLA-4 and PD-L1 mAbs, respectively, were stillalive at 90 days after intracranial injection, demonstrating

Figure3. Early blockadeofCTLA-4, PD-L1and IDO leads toprolongedT-cell–mediatedsurvival against brain tumors.A, timeline demonstrating the intracranialinjection of 4 � 105 GL261 cells followed by the intraperitoneal injection of CTLA-4, PD-L1, CD4, and/or CD8 mAbs at days 7, 10, 13, and 16 after i.c.and the oral availability of D1-MT on days 7 through 37 after i.c. B, survival analysis ofWTmice intracranially (i.c.) injectedwith 4� 105GL261 cells and treatedwith CTLA-4 mAb (clone 9H10), PD-L1 mAb (clone 10F.9G2), or both CTLA-4 and PD-L1 mAbs (n ¼ 10/group). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.C, survival analysis of WT mice i.c. injected with 4� 105 GL261 cells and treated with D1-MT, D1-MT, and CTLA-4 mAb, D1-MT and PD-L1 mAb or D1-MT,CTLA-4 and PD-L1 mAbs (n ¼ 5/group). �, P < 0.05. D, survival analysis of WT (n ¼ 10/group) or Rag1�/� (n ¼ 5–6/group) mice intracranially injected with4 � 105 GL261 cells, with or without treatment with D1-MT, CTLA-4 and PD-L1 mAbs, with or without depletion for CD4þ- and/or CD8þ-T cells.E, survival analysis of IDO�/�mice intracranially injectedwith 4� 105GL261 cells, alone (n¼4/group), or treatedwith D1-MT,CTLA-4mAband/or PD-L1mAb(n ¼ 9/group). �, P < 0.05.

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an extraordinary survival benefit in glioma. To determinethe impact of 1-MT on this approach, we analyzed micebearing glioma and administered 1-MT, alone, or in com-binationwith CTLA-4mAb, PD-L1mAb, or coadministeredwith both CTLA-4 and PD-L1 mAbs (Fig. 3C). Excitingly,100% of mice treated with the triple therapy showed dura-ble survival, when compared with only 20% ofmice treatedwith 1-MT, alone (P < 0.05). To test whether this tripleimmunotherapy required T cells to mediate the survivaleffect, we coadministered CTLA-4mAb, PD-L1mAb, and 1-MT with CD4- and/or CD8-depleting mAb(s), whichresulted in complete abrogation of any survival benefit (Fig.3D). Similarly, when we treated Rag1�/� mice with thetriple therapy, a similar lack of survival benefit wasobserved, confirming that T cells are required for thisapproach to be effective. Finally, we tested this effect inIDO�/� mice, given the recent data showing a synergisticbenefit of CTLA-4 or PD-1/PD-L1 blockade in B16-F10 cell-based peripheral tumors (31). As shown in Fig. 3E, periph-eral IDOdeficiencynegated themaximal amount of survivalbenefit, as seen in WT mice, with only 67% of mice thatreceived the triple therapy surviving to 90 days after intra-cranial injection. Collectively, these data demonstrate thatthe triple immunotherapy is highly effective at increasingsurvival in glioma-bearing mice and suggests a complex

mechanism that requires peripheral cell expression of IDOto maximize therapeutic benefits.

Triple therapy against CTLA-4, PD-L1, and IDOdecreases Tregs in glioma

Tregs (CD4þCD25þFoxP3þ) are potently immunosup-pressive T cells that infiltrate human GBM (19), suppressthe cytotoxic effector arm (32), and promote pathogen-esis in experimental brain tumor models (18, 20, 33).Therefore, depleting them directly or utilizing immuno-therapy to neutralize their presence is a major ongoinggoal for improving standard-of-care treatment forpatients with GBM. As shown in Fig. 4A, brain-residentTreg levels were decreased by treatment with triple CTLA-4, PD-L1, IDO blockade, but not by 1-MT alone, nor bycombinations of 1-MT with CTLA-4 or PD-L1 mAbsversus untreated control mice (P < 0.01). Interestingly,this effect was also seen for the frequency of Tregs expres-sing high levels of CD44 (P < 0.01), a marker of antigenexperience. In contrast, the triple therapy neither affectedthe frequency of brain-resident cytolytic CD8þ T cells(Tc), nor did it affect the level of antigen-experiencedTcs (Fig. 4B). Moreover, while the triple therapy did notaffect the levels of IFN-g expression in brain-infiltratingCD4þ T cells, when compared with control, it was

Figure 4. Early [1 week postintracranial injection (wp-i.c.)]therapeutic blockade againstCTLA-4, PD-L1 and IDOdecreasesTreg levels and increases IFN-gþ

Tcs in brain tumors. Wild-type (WT)mice intracranially injectedwith 4�105 GL261 cells and left untreatedor administered D1-MT, CTLA-4mAb (clone 9H10) and/or PD-L1mAbs (clone 10F.9G2). Thefrequency of (A) CD4þFoxP3þ andCD4þFoxP3þCD44þ Tregs, (B)CD8þ and CD8þCD44þ Tcs, or (C)CD4þIFN-gþ and CD8þIFN-gþ

effector-Tonv and -Tcs,respectively, isolated from thebrain, bilateral deep and superficialcervical draining lymph nodes(cLN) and the spleen of glioma-bearing mice at 3 wp-i.c. leftuntreated, or administeredD1-MT, CTLA-4 and/or PD-L1(n¼ 5/group). All T-cell populationswere initially identified by theexpression of CD3. �, P < 0.05;��, P < 0.01.






associated with higher IFN-g levels in Tc cells (P < 0.05).Taken together, triple blockade of CTLA-4, PD-L1, and IDOin glioma-bearing mice decreases antigen-experienced Treglevels, while coincidently increasing armed cytolytic T cells.

Given the dramatic effects of simultaneous CTLA-4, PD-L1, and IDO blockade on brain-resident Tregs and Tcs inglioma-bearing mice, we hypothesized that, in addition tothe effect on overall T-cell frequency, there would also be aneffect on the expression of immunomodulatory targets and/or receptors. Both CTLA-4 and PD-1 have previously beenshown to be potentially high value targets in experimentalmousemodels ofmalignant glioma (34, 35). Tounderstandtheir expression on T cells in the context of a responsive andproductive antitumor response, we analyzed Tregs, Tconv,and Tcs for these molecules at 3 weeks after intracranialinjection to determine whether the expression changescommensurately. As shown in Supplementary Fig. S1A,while the triple immunotherapy did not change the expres-sion of PD-1 on either Tregs or Tcs, PD-1mean fluorescenceintensity (MFI) on Tconv increased from 369 � 49 inuntreated glioma-bearing mice to 790 � 181 in mice thatreceived triple immunotherapy (P < 0.01). In contrast, thetriple immunotherapy decreased CTLA-4 expression on

Tregs from 1,527 � 176 in untreated mice to 715 � 222in mice receiving triple therapy, whereas it increased onTconv from 434 � 72 to 1,076 � 272 (P < 0.001, respec-tively; Supplementary Fig. S1B). Collectively, these datasuggest that the Tconv subset is particularly sensitive to theeffects of CTLA-4/PD-L1/IDO blockade and that as Treglevels decrease due to the effects of triple immunotherapy,the CD4þ T-cell expression for PD-1 and CTLA-4 changescommensurately.

CTLA-4/PD-L1/IDO blockade targets Tregs andenhances survival from established glioma

Since our previous observation in smaller, less estab-lished brain tumors suggested the triple therapy primarilydecreased Tregs, we wondered whether this effect would bemaintained in larger,morewell-established glioma—a timepoint that coincides with brain tumors that are � 2 mm indiameter (9). As shown in Fig. 5A and B, there was adramatic decrease in Treg levels from 38� 2% in untreatedmice to 5.3 � 1% in mice treated with triple therapy (P <0.001), which was reversed with the addition of temozo-lomide back to 39� 4% (P < 0.001). Importantly, the tripletherapy decreased Treg levels, when compared with dual

Figure 5. Late [2weeks post intracranial injection (wp-i.c.)] blockade ofCTLA-4, PD-L1, and IDOblockade decreases Treg levels and increases survival againstbrain tumors. A, timeline demonstrating the intracranial injection of 4 � 105 GL261 cells followed by the intraperitoneal injection of CTLA-4 and PD-L1mAbs at days 14, 17, 20, and 23 after i.c., the oral availability of D1-MT on days 14 through 44 after i.c. and intraperitoneal administration of TMZ ondays 14, 16, 18, as well as 21, 23, and 25 after i.c. B, the frequency of CD4þFoxP3þ Tregs and CD8þ Tcs isolated from the brain, bilateral deep andsuperficial cervical draining lymph nodes (cLN) and the spleen of tumor-bearing mice at 3 wp-ic. (n ¼ 5/group). C, survival analysis of WT mice i.c. injectedwith 4� 105 GL261 cells, alone (n¼ 7/group), or treated with (D) 1-MT, CTLA-4mAb (clone 9H10), PD-L1mAb (clone 10F.9G2) and/or TMZ (n¼ 9–10/group).D, survival analysis of IDO�/� mice i.c. injected with 4 � 105 GL261 cells, alone (n ¼ 4/group), or treated with D1-MT, CTLA-4 mAb (clone 9H10),PD-L1 mAb (clone 10F.9G2) and/or TMZ (n ¼ 7–8/group). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

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treatment with CTLA-4/PD-L1 mAbs (P < 0.05), suggestingthat all three targets require therapeutic modulation tosignificantly affect this T-cell subset in glioma. Interestingly,triple therapy also decreased brain-resident Tc levels, whencomparedwithuntreatedmice, suggesting apossible overallreduction in inflammation mediated by this approach.Importantly, this effect did not translate into differences inthe level of CD44high-, CTLA-4þ-, PD-1þ-, AiolosþCD39þ-Treg and Tc cells, although it did have an effect on the levelof BTLA expressedby tumor-infiltrating Tcs (SupplementaryFig. S2A-G). As shown in Fig. 5C, untreated WT mice had amedian overall survival of 32 days after intracranial injec-tion with 100% mortality. In contrast, 1-MT alone,increased themedian survival to 45.5 days after intracranialinjectionwith 38%ofmice alive at 90 days after intracranialinjection (P < 0.01). Incredibly, both coadministration ofCTLA-4/PD-L1 mAbs or triple therapeutic CTLA-4/PD-L1/IDO blockade resulted in durable survival for 78% ofglioma-bearing mice (P < 0.001). Not surprisingly, theconcurrent addition of temozolomide to this therapeuticregimen decreased maximal survival, suggesting that activechemotherapy in the context of productive antitumorimmunity is an undesirable approach. Interestingly, whenthe dual and triple therapies were utilized in IDO�/� micebearing glioma, maximal therapeutic efficacy was unattain-able (Fig. 5D), reinforcing our previous observation thatperipheral IDO is required for effective immunotherapy inthe context of CNS tumors. Ultimately, these data showexciting preclinical results demonstrating the efficacy ofeither dual or triple immunotherapy utilizing CTLA-4,PD-L1, and/or IDO blockade.

Intracranial melanoma is poorly responsive to CTLA-4/PD-L1/IDO blockadeGL261 cell-based brain tumors robustly recruit Tregs,

which has been shown to be one of the ways that CNSmalignancies subvert the antitumor response. However, wewere curious as to whether this was a tumor intrinsicphenomenon or whether anymalignant intracranial tumorultimately causes Treg accumulation. As shown in Fig. 6A,WT mice analyzed at 12 days after intracranial injection ofGL261 tumor possess 27% � 7% Tregs in the brain, whencompared with B16-F10 tumors that recruit only 2% �0.3% Tregs (P < 0.01). This is not a reflection of a differencein overall Treg phenotype, as Tregs isolated from both typesof intracranial tumors exhibit the same level of GITR, aprototypical receptor that is highly expressed by Tregs andknown to regulate their function in tumors (Fig. 6B; ref. 36).When the GL261 and B16-F10 cell lines were analyzed, invitro, for PD-1, GITR, and PD-L1, quantifiable differences inGITR and PD-L1were observed after stimulationwith IFN-g(Fig. 6C). However, whether these differences affect Tregaccumulation has yet to be established. To determine theeffects of our triple immunotherapeutic approach againstintracranialmelanoma,we began immunotherapy at 3 daysafter intracranial injection in WT mice given the knownaggressiveness of B16-F10 cells (Fig. 6D). To our surprise, 1-MT alone (P < 0.01), CTLA-4/PD-L1 blockade (P < 0. 01), as

well as combining all three reagents (P < 0.001) increasedoverall survival. However, the overall benefit was limited todays, rather thanmonths, aswe had observed inGL261 cell-based brain tumors. Moreover, no difference in overallsurvival was found when a similar approach was used inIDO�/�mice (Fig. 6E), nor when that approach was furthercombined with GITR and Lag-3 mAbs. These data suggestthat the efficacy of inhibiting CTLA-4, PD-L1, and IDO inbrain tumors will depend on context and based on the datawe have presented here, will be more effective in tumorsreliant on Treg accumulation, rather than aggressive tumorsknown to migrate and evade the immune response byalternative mechanisms.

DiscussionCurrent standard-of-care treatment for patients initially

diagnosed with glioblastoma multiforme (GBM) includessurgical resection, radiation, and chemotherapywith Temo-dar (temozolomide). However, this aggressive regimen canleave undesirable side effects, with an average overall sur-vival advantage of only 14.6 months after diagnosis. Thesegrimpotential outcomes have served as rationale to developalternative approaches for treating patients with high-gradeprimary brain tumors of which immunotherapy is a leadingcandidate for producing effective, durable and long-lastingpatient benefits (32, 37, 38). While these highly promisingstudies warrant further investigation, it is helpful to alsounderstand the redundant and compensatory immunosup-pressive pathways that undermine the efficacy of immuno-therapy, as well as general antitumor immunity (39, 40).Among these pathways, IDO plays a central role in regu-lating immunosuppression, as the genetic ablation of IDO,specifically in glioma cells, leads to the spontaneous andrapid rejection of brain tumors (9). Other high value targetsthat have been demonstrated to regulate immunosuppres-sion in glioma includeCTLA-4 (34), PD-L1 (41), PD-1 (35).However, to the best of our knowledge, no previous studysimultaneously targeted these regulatoryhubs in the contextof malignant glioma.

Through the mixture of IDO-competent and -deficientglioma cells, we began our investigation by asking thesimple question: which proportion of IDO-deficient cellsinduces an antitumor immune response that results in adurable survival advantage? The data indicate that micewith brain tumors composed of >50% IDO-competentglioma cells mice had a shorter overall survival, whencompared with those tumors with a composition of�50% IDO-deficient glioma cells. This observation is con-cordant with previous immunocytochemical analysis inhuman GBM, demonstrating that the majority of GBMcases are >50% positive for IDO in tumor cells (7). Inter-estingly, when IDO-competent and -deficient brain tumorswere independently established in contralateral cerebralhemispheres, the IDO-competent tumors were capable ofabrogating any survival effect normally attributable to IDO-deficient tumors. This was not simply due to the burden oftumor cells intracranially injected into the mouse brain,






since when IDO-deficient glioma cells were dually injected,the majority of mice survived and lived for up to 150 daysafter intracranial injection. Rather, this may reflect thedifference in the programming and/or recruitment of stro-mal cells that IDO-deficient glioma cells recruit to induceantitumor immunity. Importantly, the factors recruited byIDO-deficient glioma cells during the priming phase lead-ing to antitumor immunity are sufficient to cause rejectionof IDO-competent tumor cells, with a diluted level ofeffectiveness if challenged after priming has alreadyoccurred. Finally, given our previous data suggesting thatCD4þ T cells are required to reject IDO-deficient tumorsbased on the lack of long-term durable survival in CD4�/�

mice (9), we determined the relevance of antigen specificityin this survivalmechanism. As expected, OT-IImice bearing

CD4þ T cells universally antigen-restricted to chicken oval-bumin, a non-glioma expressed protein, were incapable ofrejecting both IDO-competent and -deficient brain tumors.Collectively, these data suggest a critical role for IDO insuppressing the antitumor response that depends on theproportion of IDO-expressing cells, factors differentiallyinduced/recruited depending on IDO competency, as wellas the requirement of TCR specificity to tumor and/orstromal antigens.

Given the dominant role of IDO in suppressing immu-nemediated glioma rejection, we next asked whether inhi-biting the IDO pathway via 1-MT would be sufficient torecapitulate our observations with genetic silencing. On thebasis of previous studies demonstrating that, 1-MT alone,does not lead to tumor rejection but that, 1-MT in

Figure 6. Intracranial melanoma is infiltrated by decreased Treg levels and is not amenable to durable survival with CTLA-4/PD-L1/IDO blockade. A, wild-typemice (WT) intracranially-injected (i.c.) with 4� 105 GL261- or 2–5� 103 B16-F10-cells were analyzed for Treg levels in the brain, bilateral deep and superficialcervical draining lymph nodes (cLN) and the spleen of tumor-bearing mice at 12 days post intracranial injection (dp-i.c.; n ¼ 4–6/group). B, MFIofGITRexpressiononTregs andTconvanalyzedat 12dp-i.c., asdescribed inA.C,MFI for surface expressionofPD-1,GITR, andPD-L1onGL261 (blackbars)and B16-F10 (white bars) after 24 hours in culture and stained with isotype mAb (Iso), untreated (No Tx) or treated with 10 ng/mL IFN-g (þIFN-g ).D,wild-typemicewere intracranially injectedwith 2–5�103B16-F10cells followedby the intraperitoneal injectionofCTLA-4 andPD-L1mAbsat days3, 7, 10,and 13 (when possible) after i.c., as well as the oral availability of D1-MT on days 3 through the end of the survival experiment. E, wild-type mice wereintracranially injected with 2–5� 103 B16-F10 cells followed by intraperitoneal injection of CTLA-4 and PD-L1 mAbs at days 3, 7, 10, and 13 (when possible)after i.c., as well as the oral availability of D1-MT on days 3 through the end of the survival experiment. Survival analysis of WT mice i.c. injected with4 � 105 GL261 cells, alone (n ¼ 7/group), or treated with D1-MT, CTLA-4 mAb (clone 9H10), PD-L1 mAb (clone 10F.9G2), and/or TMZ (n ¼ 9–10/group).Bar graphs, mean � SEM. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Wainwright et al.





combinationwith chemotherapy leads to productive tumorimmunity (10), we chose to study the treatment of gliomawith 1-MT in combination with temozolomide. We foundthat both levorotary (L) and dextrorotary (D) stereoisomersof 1-MT were ineffective at increasing overall survival fromglioma burden when administered at 2 mg/mL in thedrinking water. However, when both D1-MT and L1-MTwere combined at 5 mg/mL they showed a significantantitumor effect, possibly reflecting a higher dosing require-ment due to poor blood–brain barrier permeability. Inter-estingly, when either L1-MT orD1-MTwere coadministeredwith temozolomide under the dosing regimen used in thisstudy, neither combinatorial regimen increased survivalwhen compared with mice treated with temozolomidealone. This suggests that, in contrast with peripheral tumormodels, the synergismbetween IDO inhibition and glioma-induced death may differ in sensitivity toward this thera-peutic regimen. Alternatively, the lack of synergistic antitu-mor effect could be due to repeated and frequent dosingwith temozolomide, whichmay abrogate the establishmentof a productive immune response. Another possible expla-nation includes compensation due to kynurenine produc-tion by IDO2and/or TDO. In support of this hypothesis, wefound that all three mammalian tryptophan catabolicenzymes, IDO1, IDO2, and TDO, were expressed in braintumors. Recent evidence suggests that both IDO2 and TDOcontribute to the regulation of immunity (42) and/orprogression of glioma (43).Given that 1-MT and temozolomide did not produce

beneficial results when compared to temozolomide, alone,we hypothesized that coupling IDO inhibition with theregulation of other powerful immunomodulatorymediatorswould produce a synergistic survival response in glioma-bearingmice.Data fromperipheral tumormodels previouslydemonstrated that combining CTLA-4 and PD-1 and/or PD-L1 inhibition is an attractive method for reducing immuno-suppression and/or reactivating productive antitumorresponse (44–46). On the basis of this literature, we won-dered whether this approachwould also yield benefits in thecontext of aggressive neoplasms within the central nervoussystem (CNS), a site normally considered to be relativelyimmune privileged. Much to our surprise, the simultaneoustherapeutic inhibition of CTLA-4 and PD-L1 at 1 weekfollowing glioma cell implantation led to a remarkably highsurvival rate of 90% in glioma-bearing mice. Notably, thissurvivalwas durableover a 90-dayperiodofobservation. Theaddition of 1-MT to CTLA-4 and PD-L1 blockade was alsoassociated with a high survival rate of 100% in glioma-bearing mice. Notably, the survival benefit induced by tripleimmunotherapy was completed abrogated when theimmune systemwas depleted for CD4þ and/orCD8þ T cells.This implies that there is a coordinatedmechanism of actionbetween both T-cell subsets to carry out effective gliomaimmunity. However, the temporal requirement after thera-peutic initiation for eachT-cell subtype,whichcells theymustinteract with in the tumor microenvironment and whetherthose interactions mediate direct or indirect antitumoreffects, have yet to be determined.

One unexpected finding from our study was that CTLA-4,PD-L1� IDOblockade inmice deficient for peripheral IDO(i.e., non-glioma derived) showed decreased overall surviv-al, when compared with WT mice. This is a somewhatparadoxical observation given the recent finding thathost-derived IDO plays a critical role in antitumor immu-nity when coupled with either CTLA-4 or PD-1 blockade(31). However, it is important to keep in mind that what isoften observed in the CNS, regardless of the presence orabsence of neoplasm, does not recapitulate an identicalresponse, peripherally. This is likely due, in part, to thepresence of the blood–brain barrier, lack of a developedlymphatic system, the normally low expression of MHCclass II, as well as the different stromal cells within the CNSparenchyma.

We found that, regardless of early or late blockade forCTLA-4, PD-L1, and IDO,Treg levelswere overall decreased.Interestingly, the late administration of immunotherapyalso induced a decrease in brain tumor-infiltrating Tcs. Thislatter observation has potentially important implicationsrelated to the overall inflammatory state in the brain.Theoretically, any immunotherapy will generate somedegree of inflammation associated with the production ofproinflammatory cytokines and cell death associated withtumor rejection. Thus, a therapy that is effective for killingtumor cells, but recruits fewer leukocytes to the CNS, ishighly desirable. This is particularly notable since the com-mon way to decrease inflammation in the CNS for patientswith GBM is by administering Decadron, a glucocorticoidthat will likely marginalize an active T-cell–mediatedresponse. Thus, while overall survival is similar betweendual and triple therapeutic approaches, the additionaladvantage of decreased inflammatory cell infiltration war-rants further investigation.

Our investigation found that CTLA-4/PD-L1 mAb � 1-MT treatment in the context of intracranially injectedGL261(glioma) cell-based brain tumors resulted in a highly effec-tive and durable survival advantage, while the intracraniallyinjected B16-F10 (melanoma) tumor model, showed adramatically reduced level of efficacy. Other notable differ-ences included a significantly decreased level of Treg infil-tration in B16-F10 cell-based tumors with higher levels ofIFN-g–inducible GITR and B7-H1 expression. Interestingly,there was no difference in survival when B16-F10 cells wereintracranially injected in IDO�/� mice, again highlightingthe contextual difference between eradicating peripheraltumors as previously shown (31) with the data we haveincluded here. Given the capability that both GL261 andB16-F10 cells express IDO and consequently modulateantitumor immunity (9, 47), it will be interesting to deter-mine whether both types of tumors express similar levels ofIDO, IDO2, and TDO.

In summary,we have extended our previous observationsdelineating the impact of IDO in brain tumors, demon-strated that all three mammalian tryptophan catabolicenzymes are present and have found a potent method toinduce the rejection of primary tumors by virtue of CTLA-4and PD-L1 blockade. By coupling this therapeutic regimen






with 1-MT, we were able to reduce Treg and Tc levels inestablished brain tumors with similar levels of overallsurvival and durable efficacy. Since clinical-grade analogsare available for all three agents that we tested, this strategyhas high therapeutic value for patients with GBM. Whetherthis approachwill be equally effective for those patients thatare initially diagnosed versus those who present with recur-rent tumors has yet to be explored and is difficult to modelexperimentally. Also, our data suggests that concomitantadministration of CTLA-4/PD-L1/IDO blockade and temo-zolomide is not advantageous. Accordingly, we plan todetermine whether staggering the treatments avoids thera-peutic abrogation in the future. Ultimately, this work servesa proof-of-concept that this type of approach works and isrelevant for treating patients with incurable malignantglioma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D.A. Wainwright, M. Dey, I.V. Balyasnikova,J.W. Kim, J. Qiao, M.S. LesniakDevelopment of methodology: D.A. Wainwright, J.W. Kim, M.S. Lesniak

Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): D.A. Wainwright, A.L. Chang, M. Dey, C.K. Kim,A. Tobias, Y. Cheng, M.S. LesniakAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):D.A.Wainwright, A.L. Chang, I.V. Balyas-nikova, J.W. Kim, L. Zhang, M.S. LesniakWriting, review, and/or revision of the manuscript: D.A. Wainwright,M. Dey, I.V. Balyasnikova, J.W. Kim, M.S. LesniakAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.A. Wainwright, Y. Han,M.S. LesniakStudy supervision: D.A. Wainwright, M.S. Lesniak

AcknowledgmentsThe authors thank Dr. Mario R. Mautino for making significant intellec-

tual contributions toward this project and article.

Grant SupportThis work was supported by an American Brain Tumor Association

Discovery Grant (to D.A. Wainwright), as well as NIH grants NIH F32NS073366 (toD.A.Wainwright), NIHK99NS082381 (toD.A.Wainwright),R01CA138587 (toM.S. Lesniak),NIHR01CA122930 (toM.S. Lesniak), andNIH U01 NS069997 (to M.S. Lesniak).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 28, 2014; revised March 24, 2014; accepted March 24,2014; published OnlineFirst April 1, 2014.

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Correction

Correction: Durable Therapeutic EfficacyUtilizingCombinatorialBlockadeagainst IDO,CTLA-4, and PD-L1 in Mice with Brain Tumors

In this article (Clin Cancer Res 2014;20:5290–301), which was published in theOctober 15, 2014, issue of Clinical Cancer Research (1), the phrase ". . .whereas two-tailed unpaired Student t tests were used for paired groups," within a sentence in thestatistical analysis section, generated confusion among readers. The authors haveprovided clarification, and the new paragraph should read as follows:Datawere analyzed usingPrism4.0 software (GraphPad Software). Experimentswererepeated at least two times each. Data are represented as themean� SEM for allfigurepanels in which error bars are shown. The P values represent ANOVA for groups of3 or more, whereas two-tailed unpaired Student t tests were used for comparisonsbetween two groups. A P value of less than 0.05 was considered statisticallysignificant.The authors regret this error.

Reference1. Wainwright DA, Chang AL, Dey M, Balyasnikova IV, Kim CK, Tobias A, et al. Durable therapeutic

efficacy utilizing combinatorial blockade against IDO, CTLA-4, and PD-L1 in mice with braintumors. Clin Cancer Res 2014;20:5290–301.

Published online February 2, 2015.doi: 10.1158/1078-0432.CCR-14-3211�2015 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 21(3) February 1, 2015662

2014;20:5290-5301. Published OnlineFirst April 1, 2014.Clin Cancer Res Derek A. Wainwright, Alan L. Chang, Mahua Dey, et al. against IDO, CTLA-4, and PD-L1 in Mice with Brain TumorsDurable Therapeutic Efficacy Utilizing Combinatorial Blockade

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